Dictyostelium diccoideum life cycle and cloning

1. Summer Training Presentations
Department of Molecular and Human Genetics

2. Understanding of the life cycle of Dictyostelium
discoideum and cloning of putative gene
Mentor:
Dr. Shweta Saran
Developmental Biology Lab
SLS JNU, New Delhi.
Presented By:
Bhupender Verma
M.Sc. (F) MHG BHU

3. Introduction
 Dictyostelium discoideum are single
cellular organisms
Live on decaying logs and feed on
bacteria
Dictyostelium discoideum follow asexual
cycle-
• when there is plenty of food available
• Live solitary
• Are haploid
• Reproduce by binary fission
 Also called as “Social amoebae”
During starvation organisms aggregate
and migrating slug is formed that
culminates in a fruiting body to release
spores

4. Figure: Scott F. Gilbert-Developmental Biology 8th edition

5. Classification:
Domain – Eukarya
Kingdom- Amoebozoa
Superphylum- Conosa
Phylum- Mycetozoa
Class- Dictyostelia
Order- dictyosteliida
Family- Dictyosteliidae
Genus- Dictyostelium
Species- D. discoideum

6. Life cycle
 In lab Dicty are grown in HL5 liquid media at 22ºC.
 In nutrient rich conditions Dicty divide by binary fission
and remain solitary.
 For studying their development a concentrated solution
is transferred to the NNA plates.
 In the absence of nutrients, these myxomoebae join
together to form moving streams of cells converging at a
central point.
 Different developmental stages that can be observed:
• Tight aggregate
• Migrating slug (pseudoplasmodium or grex) – slimy
sheath encased
• Fruiting body

7. Vegetative loose aggregate mound migratory slug
Early culminant fruiting body

8. Cloning of the putative gene:
Strategy:
• Design the primers
• Gradient PCR for optimizing annealing conditions
• Standard PCR for obtaining amplified insert
• Digestion of vector and insert with HindIII and PstI
• Liagtion of insert to the vector
• Transform bacteria with recombinant vector
• Isolate cloned plasmid and digest for checking if insert is
desirable

9. Gradient PCR for optimizing annealing conditions:
1 2 3 4 5 6 7 8 9 10 M 11 12 13 14 15 16 17 18 19 20 Design the primers
• Gradient PCR
5 kb • Standard PCR
1.5 kb • Digestion with HindIII
500 bp and PstI
• Liagtion
• Bacterial
21 22 23 24 25 26 27 28 29 30 M 31 32 33 34 35 36
transformaton
• Isolate cloned plasmid
5 kb
and digest for checking
1.5 kb if insert is desirable
500 bp

10. Standard PCR for obtaining amplified insert:
Design the primers
• Gradient PCR
• Standard PCR
• Digestion with HindIII
5 kb and PstI
• Liagtion
1.5 kb • Bacterial
transformaton
500 bp • Isolate cloned plasmid
and digest for checking
if insert is desirable

11. pDrive cloning vector-
Design the primers
• Gradient PCR
• Standard PCR
• Digestion with HindIII
and PstI
• Liagtion
• Bacterial
transformaton
• Isolate cloned plasmid
and digest for checking
if insert is desirable

12. Digestion of vector and insert with HindIII and PstI
Design the primers
• Gradient PCR
• Standard PCR
• Digestion with HindIII
and PstI
5 kb • Liagtion
• Bacterial
1.5 kb transformaton
• Isolate cloned plasmid
and digest for checking
500 bp
if insert is desirable

13. Checking insert from fallout size:
Design the primers
5 kb • Gradient PCR
1.5 kb • Standard PCR
500 bp • Digestion with HindIII
and PstI
• Liagtion
• Bacterial
transformaton
5 kb
• Isolate cloned plasmid
1.5 kb
and digest for checking
500 bp if insert is desirable

14. Cloning two segments of a putative gene
in pDrive vector for making Knockout-
1. Insert 1 – obtained from digestion (HindIII & NotI) of cloning
vector as pop out
2. pDrive vector – already had other insert so digest with (HindIII &
NotI) & obtain vector-BSR
3. Ligate insert to vector
4. Transform bacteria
5. Grow bacteria and isolate plasmid
6. Digest plasmid for checking proper ligation
7. Proceed with the colony that had proper insert
8. PCR of insert 2
9. Digest insert 2
10.Ligation
11.Transformation
12.Isolate plasmid and check

15. 1. Insert 1-as pop out
Digestion with HindIII & NotI 2. pDrive -had other
insert
3. Ligation of insert 1
4. Transform E.coli
5. Grow E.coli &
isolate plasmid
6. Digest plasmid for
check
7. Proceed with sample
+BSR having proper insert
8. PCR of insert 2
9. Digest insert 2
10.Ligation
11.Transformation
12.Isolate plasmid and
check

16. 1. Insert 1-as pop out
Check after elution from 2. pDrive -had other
agarose gel: insert
3. Ligation of insert 1
4. Transform E.coli
5. Grow E.coli &
isolate plasmid
6. Digest plasmid for
5 kb
check
7. Proceed with sample
1.5 kb having proper insert
8. PCR of insert 2
500 bp 9. Digest insert 2
10.Ligation
11.Transformation
12.Isolate plasmid and
check

17. 1. Insert 1-as pop out
For checking again digest 2. pDrive -had other
with HindII & NotI insert
3. Ligation of insert 1
4. Transform E.coli
5 kb 5. Grow E.coli &
1.5 kb isolate plasmid
500 bp 6. Digest plasmid for
check
7. Proceed with sample
having proper insert
8. PCR of insert 2
5 kb 9. Digest insert 2
1.5 kb 10.Ligation
500 bp 11.Transformation
12.Isolate plasmid and
check

18. 1. Insert 1-as pop out
For checking again digest 2. pDrive -had other
with HindII & NotI insert
3. Ligation of insert 1
4. Transform E.coli
5 kb 5. Grow E.coli &
1.5 kb isolate plasmid
500 bp 6. Digest plasmid for
check
7. Proceed with sample
having proper insert
8. PCR of insert 2
5 kb 9. Digest insert 2
1.5 kb 10.Ligation
500 bp 11.Transformation
12.Isolate plasmid and
check

19. After insert1 what do we have?
pDrive-Insert1-BSR

20. 1. Insert 1-as pop out
Digestion with HindIII & 2. pDrive -had other
BamhI: insert
3. Ligation of insert 1
4. Transform E.coli
5. Grow E.coli &
isolate plasmid
6. Digest plasmid for
check
7. Proceed with sample
5 kb having proper insert
1.5 kb 8. PCR of insert 2
500 bp 9. Digestion of insert 2
10.Ligation
11.Transformation
12.Isolate plasmid and
check

21. 1. Insert 1-as pop out
Result: 2. pDrive -had other
insert
3. Ligation of insert 1
4. Transform E.coli
5. Grow E.coli &
pDrive Insert1-BSR-Insert2
isolate plasmid
6. Digest plasmid for
check
7. Proceed with sample
Now we are ready with a DNA having proper insert
8. PCR of insert 2
segment that is “insert1-BSR-
9. Digestion of insert 2
insert2”
10.Ligation
This insert can be used for 11.Transformation
knocking out this putative genes 12.Isolate plasmid and
And my insert is still under study check

22. References:
RICARDO ESCALANTE and JUAN J. VICENTE- Dictyostelium
discoideum: a model system for differentiation and patterning(2000)
L. Eichinger, J. A. Pachebat, G. Glockner- The genome of the social
amoeba Dictyostelium discoideum (2005)
Developmental biology- Scott F. Gilbert- 8th edition.

23. Thank you!